PAPER-BASED CELLULAR ARRAYS
Three-dimensional cellular arrays, methods of making three-dimensional cellular arrays, and methods of identifying agents using the arrays are disclosed.
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This application claims the benefit of U.S. Provisional Application No. 61/040,030, filed Mar. 27, 2008; U.S. Provisional Application No. 61/040,010, filed Mar. 27, 2008; U.S. Provisional Application No. 61/097,718, filed Sep. 17, 2008; and U.S. Provisional Application No. 61/146,413, filed Jan. 22, 2009; the contents of all of which are hereby incorporated in their entirety herein.
BACKGROUNDCells in vivo reside in an organized three-dimensional environment as part of tissue and organ structures; loss of this tissue organization is a hallmark of cancer (Wodarz, et al., Nature Cell Biology 9:1016-1024 (2007); Lee, et al., J. Cell Sci. 121:1141-1150 (2008); Morrison, et al., Nature 441:1068-1074 (2006)). For nearly a century, progress in cancer research and discovery of anti-cancer agents have been fueled by investigations performed on cells cultured outside the living organism (ex vivo) (Ebeling, J. Exp. Med. 17:273-285 (1913); Carrel, et al., J. Exp. Med. 13:387-U34 (1911); Carrel, et al., J. Exp. Med. 13:571-575 (1911); Leighton, Cancer Res. 17:929-941 (1957); Paul, Cancer Res. 22:431-& (1962)). The majority of these studies have been performed with cells cultured on two dimensional surfaces. Morphological and functional differences of cells cultured in these conditions and cells in vivo have been widely recognized, and three-dimensional cell growth substrates have been shown to present a more physiologically relevant model of in vivo cell environment (Yamada, et al., Cell 130:601-610 (2007); Nelson, et al., Annu. Rev. Cell Dev. Biol. 22:287-309 (2006); Huang, et al., Nature cell biology 1:E131-E138 (1999); Schmeichel, et al., J. Cell Sci. 116, 2377-2388 (2003)). Importantly, cells cultured on two-dimensional substrates often do not respond to soluble factors that influence cells in three-dimensional environments (Emerman, et al., In Vitro-Journal of the Tissue Culture Association 13:316-328 (1977); Emerman, et al., Proc. Natl. Acad. Sci. U. S. A. 74:4466-4470 (1977); Cukierman, et al., Science 294:1708-1712 (2001); Bissell, et al., Differentiation 70:537-546 (2002); Weaver, et al., J. Cell Biol. 137:231-245 (1997)). Yet, to date, the majority of drug discovery processes start from small-molecule screening in two-dimensional culture-based assays. Failures of the identified compounds in animal and human trials drive the cost of drug discovery to >$1 billion per new compound (Griffith, et al., Nat. Rev. Mol. Cell Biol. 7:211-224 (2006)). High-throughput assays based on three-dimensional cultures can allow assessment of drug efficiency and toxicity at the very first step of the drug discovery process. Integration of the three-dimensional cell culture into every aspect of basic and applied cancer research will advance the discovery of new therapeutics for cancer treatment.
Differences in cell responses in three-dimensional vs. two-dimensional environment originate from differences in cell polarity, cellwide distribution of the substrate adhesion sites and responses of cells to mechanical properties of the matrix (Yamada, et al,. 2007; Huang, et al., 1999). On the molecular level, these events are regulated by the cross-talk between integrin signaling pathways and those of the receptor-tyrosine kinases. Properties like chemical composition of the matrix, nano- and microscale distribution of the integrin ligands (Cukierman, et al., 2001; Chen, et al., Science 276:1425-1428 (1997)), as well as mechanical property of the matrix (Engler, et al., Cell 126:677-689 (2006); Pelham, et al., Proc. Natl. Acad. Sci. U. S. A. 94:13661-13665 (1997); Yeung, et al., Cell Motil. Cytoskeleton 60:24-34 (2005)), can influence these pathways and modulate cell behavior. Furthermore, delivery of oxygen and nutrients to cells in gel-like matrix is driven by diffusion; hence, physical dimensions of the matrix also play a role in three-dimensional cell culture. Due to diffusion limitations, proliferation of cells in three-dimensional matrices ex vivo is often limited to a depth of less than a few hundred microns. Therefore, the size, composition, and mechanical properties of the matrix must be carefully controlled in three-dimensional culture.
After decades of side-by-side development of two-dimensional and three-dimensional culture, the simplicity of two-dimensional culture approach makes it a dominant technology for ex vivo investigation of cells. The need to control multiple chemical and physical properties of the matrix makes three-dimensional culture of cells more labor intensive and less reproducible.
SUMMARYIn one aspect, the invention features a three-dimensional cellular array. The cellular array includes a porous, hydrophilic substrate comprising a plurality of porous regions, each porous region bounded at least in part by a liquid impervious boundary; and a hydrogel comprising cells, wherein the hydrogel is embedded within the porous regions. In one embodiment, the substrate is paper, nitrocellulose, cellulose acetate, cloth, or porous polymer film.
In one embodiment, the hydrogel is a temperature sensitive hydrogel. In particular embodiments, the temperature-sensitive hydrogel is Matrigel or collagen. In some embodiments, the hydrogel is an ionotropic hydrogel. In particular embodiments, the ionotropic hydrogel comprises alginic acid (AA), carboxymethylcellulose (CMC), ι-carrageenan, poly(galacturonic acid) (PG), poly(bis(4-carboxyphenoxy)-phosphazene, or PuraMatrix.
In yet other embodiments, the liquid impervious boundary comprises PDMS, poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, PMMA, polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax or a fat.
In certain embodiments, the array comprises 1, 2, 3, 4, 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 1,000 or more porous regions, each bound by a liquid impervious boundary. In particular embodiments, the array comprises 96, 384, 1536, or 3456 porous regions, each bound by a liquid impervious boundary.
In some embodiments, the cells are bacterial cells, insect cells, yeast cells, or mammalian cells.
In another aspect, the invention features a method of making a three-dimensional cellular array. The method comprises providing a porous, hydrophilic substrate, wherein the substrate comprises a plurality of porous regions, each porous region bounded at least in part by a liquid impervious boundary; and contacting the porous, hydrophilic substrate with a suspension of cells and a temperature-sensitive hydrogel or an ionotropic hydrogel precursor, wherein the suspension saturates one or more porous regions of the substrate. In one embodiment, the substrate is paper, nitrocellulose, cellulose acetate, cloth, or porous polymer film.
In one embodiment, the hydrogel is a temperature sensitive hydrogel. In particular embodiments, the temperature-sensitive hydrogel is Matrigel or collagen. In some embodiments, the hydrogel is an ionotropic hydrogel. In particular embodiments, the ionotropic hydrogel comprises alginic acid (AA), carboxymethylcellulose (CMC), ι-carrageenan, poly(galacturonic acid) (PG), poly(bis(4-carboxyphenoxy)-phosphazene, or PuraMatrix.
In one embodiment, the method further comprising wetting, e.g., saturating, the substrate with a gelling agent before contacting the suspension of cells with the substrate. In some embodiments, the gelling agent is a metallic ion. In particular embodiments, the gelling agent is Pb2+, Ba2+, Fe3+, Al3+, Cu2+, Cd2+, Ho3+, Ca2+, Zn2+, Co2+, Ni2+, Mn2+, or Mg2.
In some embodiments, the liquid impervious boundary comprises PDMS, poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, PMMA, polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax or a fat.
In certain embodiments, the array comprises 1, 2, 3, 4, 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 1,000 or more porous regions, each bound by a liquid impervious boundary. In particular embodiments, the array comprises 96, 384, 1536, or 3456 porous regions, each bound by a liquid impervious boundary.
In some embodiments, the cells are bacterial cells, insect cells, yeast cells, or mammalian cells.
In another aspect, the invention features a method of making a three-dimensional cellular array. The method comprises providing a porous, hydrophilic substrate; contacting a plurality of defined regions of the substrate with a suspension, the suspension comprising cells and a temperature-sensitive hydrogel or an ionotropic hydrogel precursor, wherein the suspension saturates the plurality of defined regions of the substrate; and contacting the temperature-sensitive hydrogel or the ionotropic hydrogel precursor with a gelling agent, wherein the gelling agent induces the formation of a hydrogel embedded in the plurality of defined regions of the substrate. In one embodiment, the substrate is paper, nitrocellulose, cellulose acetate, cloth, or porous polymer film.
In one embodiment, the hydrogel is a temperature sensitive hydrogel. In particular embodiments, the temperature-sensitive hydrogel is Matrigel or collagen. In some embodiments, the hydrogel is an ionotropic hydrogel. In particular embodiments, the ionotropic hydrogel comprises alginic acid (AA), carboxymethylcellulose (CMC), ι-carrageenan, poly(galacturonic acid) (PG), poly(bis(4-carboxyphenoxy)-phosphazene, or PuraMatrix.
In one embodiment, the method further comprising wetting, e.g., saturating, the substrate with a gelling agent before contacting the suspension of cells with the substrate. In some embodiments, the gelling agent is a metallic ion. In particular embodiments, the gelling agent is Pb2+, Ba2+, Fe3+, Al3+, Cu2+, Cd2+, Ho3+, Ca2+, Zn2+, Co2+, Ni2+, Mn2+, or Mg2.
In some embodiments, the cells are bacterial cells, insect cells, yeast cells, or mammalian cells. In some embodiments, the method further comprising contacting the array with a culture medium.
In another aspect, the invention features a method of identifying an agent that modifies cellular function. The method comprises providing an array described herein; contacting the array with one or more test agents; and detecting one or more cellular functions in the presence of the one or more test agents; wherein a change in cellular function in the presence of the one or more test agents indicates the one or more test agents modify cellular function.
In some embodiments, the array includes a porous, hydrophilic substrate comprising a plurality of porous regions, each porous region bounded at least in part by a liquid impervious boundary; and a hydrogel comprising cells, wherein the hydrogel is embedded within the porous regions. In one embodiment, the substrate is paper, nitrocellulose, cellulose acetate, cloth, or porous polymer film.
In one embodiment, the hydrogel is a temperature sensitive hydrogel. In particular embodiments, the temperature-sensitive hydrogel is Matrigel or collagen. In some embodiments, the hydrogel is an ionotropic hydrogel. In particular embodiments, the ionotropic hydrogel comprises alginic acid (AA), carboxymethylcellulose (CMC), ι-carrageenan, poly(galacturonic acid) (PG), poly(bis(4-carboxyphenoxy)-phosphazene, or PuraMatrix.
In yet other embodiments, the liquid impervious boundary comprises PDMS, poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, PMMA, polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax or a fat.
In certain embodiments, the array comprises 1, 2, 3, 4, 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 1,000 or more porous regions, each bound by a liquid impervious boundary. In particular embodiments, the array comprises 96, 384, 1536, or 3456 porous regions, each bound by a liquid impervious boundary.
In some embodiments, the array is contacted with the one or more test agents at a plurality of porous regions. In particular embodiments, the array is contacted with the one or more test agents at 96, 384, 1536, or 3456 porous regions. In some embodiments, each porous region is contacted with a different test agent.
In some embodiments, the test agent is a small organic or inorganic molecule, an amino acid, a polypeptide, a nucleic acid, a peptide nucleic acid, a carbohydrate, or a polysaccharide. In some embodiments, the test agent is a member of a library of test agents, e.g., a combinatorial chemical library.
In other embodiments, the cellular function is proliferation, migration, viability, or gene transcription.
In another aspect, the invention features a method of identifying an agent that modifies cellular function. The method comprises providing a three-dimensional array described herein; cutting the substrate into a plurality of segments, each segment having equal dimensions; contacting each segment with a test agent or a control; and detecting one or more cellular functions in the presence of the test agent; wherein a change in cellular function in the presence of the test agent indicates the test agent modifies cellular function. In some embodiments, the cellular function is proliferation, migration, viability, or gene transcription.
In some embodiments, each segment is placed in a well of a 96-well, 384-well, 1536-well, or 3456-well plate. In some embodiments, each well contains a different test agent. In some embodiments, the test agent is a small organic or inorganic molecule, an amino acid, a polypeptide, a nucleic acid, a peptide nucleic acid, a carbohydrate, or a polysaccharide. In some embodiments, the test agent is a member of a library of test agents, e.g., a combinatorial chemical library.
In some embodiments, the cells are bacterial cells, insect cells, yeast cells, or mammalian cells.
In another aspect, the invention features a three-dimensional microarray. The microarray comprises a bottomless microtiter plate having a plurality of wells; and a porous hydrophilic substrate comprising a plurality of porous regions and a plurality of liquid impervious boundaries, each porous region bounded by a liquid impervious boundary; wherein the wells and the liquid impervious boundaries are arranged in identical patterns, the microtiter plate and the substrate attached so that the plurality of wells are aligned and sealingly joined to the plurality of liquid impervious boundaries to form an individual chamber for each porous region. In some embodiments, the porous regions of the substrate comprise cells in a hydrogel.
In one embodiment, the hydrogel is a temperature sensitive hydrogel. In particular embodiments, the temperature-sensitive hydrogel is Matrigel or collagen. In some embodiments, the hydrogel is an ionotropic hydrogel. In particular embodiments, the ionotropic hydrogel comprises alginic acid (AA), carboxymethylcellulose (CMC), ι-carrageenan, poly(galacturonic acid) (PG), poly(bis(4-carboxyphenoxy)-phosphazene, or PuraMatrix.
In yet other embodiments, the liquid impervious boundary comprises PDMS, poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, PMMA, polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax or a fat.
In certain embodiments, the array comprises 1, 2, 3, 4, 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 1,000 or more porous regions, each bound by a liquid impervious boundary. In particular embodiments, the array comprises 96, 384, 1536, or 3456 porous regions, each bound by a liquid impervious boundary.
In some embodiments, the cells are bacterial cells, insect cells, yeast cells, or mammalian cells.
In another aspect, the invention features a method of identifying an agent that modifies cellular function. The method comprises providing a microarray comprising a bottomless microtiter plate having a plurality of wells; and a porous flexible substrate comprising a plurality of porous regions and a plurality of liquid impervious boundaries, each porous region bounded by a liquid impervious boundary; wherein the wells and the liquid impervious boundaries are arranged in identical patterns, the microtiter plate and the substrate attached so that the plurality of wells are aligned and sealingly joined to the plurality of liquid impervious boundaries to form an individual chamber for each porous region; contacting the array with one or more test agents; and detecting one or more cellular functions in the presence of the one or more test agents; wherein a change in cellular function in the presence of the one or more test agents indicates the one or more test agents modify cellular function.
In some embodiments, the test agent is a small organic or inorganic molecule, an amino acid, a polypeptide, a nucleic acid, a peptide nucleic acid, a carbohydrate, or a polysaccharide. In some embodiments, the test agent is a member of a library of test agents, e.g., a combinatorial chemical library. In some embodiments, each well contains a different test agent.
In another aspect, the invention features a method of patterning a porous, hydrophobic substrate. The method comprises contacting a porous, hydrophobic substrate with an aqueous solution comprising a water-soluble compound, the solution infiltrating the substrate to form a first region of the substrate that is saturated with the solution and a second region that is not contacted with the solution; contacting the substrate with a hydrophobic material, the hydrophobic material saturating the second region; and removing the water-soluble compound, resulting in a hydrophilic porous region that is bounded by the hydrophobic material.
In some embodiments, the water-soluble compound is sucrose, trehalose, glucose, fructose, xylitol, ribose, threitol, mannose, or glycerol. In some embodiments, the aqueous solution is spotted, printed, drawn, or stamped onto the porous, hydrophobic substrate. In particular embodiments, the solution is printed using an inkjet printer.
In some other embodiments, the hydrophobic material comprises PDMS, poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, PMMA, polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax or a fat.
In certain embodiments, the substrate is patterned into 1, 2, 3, 4, 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 1,000 or more porous regions, each bound by the hydrophobic material. In particular embodiments, the substrate is patterned into 96, 384, 1536, or 3456 porous regions, each bound by a liquid impervious boundary.
In some embodiments, the substrate is nitrocellulose, cellulose acetate, cellulosic paper, filter paper, cloth, or a porous polymer film.
In another aspect, the invention features a method of patterning a porous, hydrophobic substrate. The method comprises contacting a porous, hydrophobic substrate with a hydrophobic material, the hydrophobic material saturating the substrate; contacting a region of the substrate with an aqueous solution comprising a water-soluble compound, the solution displacing the hydrophobic material from the region; and removing the water-soluble compound, resulting in a hydrophilic porous region that is bounded by the hydrophobic material.
In some embodiments, the water-soluble compound is sucrose, trehalose, glucose, fructose, xylitol, ribose, threitol, mannose, or glycerol. In some embodiments, the aqueous solution is spotted, printed, drawn, or stamped onto the porous, hydrophobic substrate. In particular embodiments, the solution is printed using an inkjet printer.
In some other embodiments, the hydrophobic material comprises PDMS, poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, PMMA, polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax or a fat.
In certain embodiments, the substrate is patterned into 1, 2, 3, 4, 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 1,000 or more porous regions, each bound by the hydrophobic material. In particular embodiments, the substrate is patterned into 96, 384, 1536, or 3456 porous regions, each bound by a liquid impervious boundary.
In some embodiments, the substrate is nitrocellulose, cellulose acetate, cellulosic paper, filter paper, cloth, or a porous polymer film.
In some aspects, an array described herein can include cells embedded within a porous, hydrophilic substrate in the absence of a hydrogel.
The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
GeneralThe invention is based, at least in part, on the production and use of three-dimensional paper-based cellular arrays. The use of micro-fabricated substrates for the generation of three-dimensional cell cultures of uniform characteristic is difficult to adopt universally, as many cell biology research groups lack the expertise or the equipment used for micro-fabrication (e.g., a clean room). In addition, many microfabrication techniques are well-suited for investigating small numbers of cells, but they are difficult to adopt for large scale assays and screens. Breakthroughs in three-dimensional cell-based assays hinge on simple and scalable techniques that allow for control of all desired properties of the three-dimensional matrix. The methods described herein can be used to produce such three-dimensional matrices.
Three-Dimensional Cellular ArraysIn certain instances, the present disclosure provides three-dimensional cellular arrays capable of growing and maintaining cells. The cellular arrays include a porous flexible substrate and a cell-containing hydrogel embedded within the substrate. Exemplary three-dimensional cellular arrays are illustrated schematically in
As described herein, the cellular array can be made by contacting, e.g., spotting, the substrate with a suspension of cells in a hydrogel or hydrogel precursor, as illustrated in
When the substrate is patterned, e.g., contains hydrophilic and hydrophobic areas, as is illustrated by substrate 1340 in
After contacting the substrate with a suspension of cells and a hydrogel or hydrogel precursor, the substrate is maintained under suitable conditions that allow gelation of the hydrogel within the substrate. As described herein, suitable conditions include maintaining the substrate at a particular temperature or contacting the substrate with a gelling agent. The resulting three-dimensional cellular array is stable and can be maintained in conditions suitable for cell growth. Such culture conditions are known in the art (see, e.g., Culture of Animal Cells: A Manual of Basic Techniques, Freshney, R. I. ed., (Alan R. Liss & Co., New York 1987); Animal Cell Culture: A Practical Approach, Freshney, R. I. ed., (IRL Press, Oxford, England 1986)). For example, the cellular array can be immersed in cell culture medium suitable for a particular cell type and maintained in an incubator.
Controlling the spotted volume allowed the regulation of the lateral dimension of the cell-filled areas. As shown in
The cellular arrays described herein can be fabricated from porous, hydrophilic substrates. In some embodiments, the substrate is paper, such as chromatographic paper. However, any substrate that wicks fluids by capillary action can be used, including, but not limited to, nitrocellulose and cellulose acetate, cellulosic paper, filter paper, cloth, and porous polymer film.
Many physical parameters of paper make it an attractive candidate for supporting the three-dimensional culture of cells: (1) Paper is an inexpensive, non-toxic, inert porous matrix; (2) flat paper sheets of well-defined thickness (>20 μm) are readily available world-wide; (3) paper can be patterned into hydrophilic and hydrophobic areas; aqueous solutions readily adopt the dimensions of the hydrophilic areas; (4) mechanical properties of the paper can be varied; and (5) paper can be easily shaped, layered or folded into different forms. Based on these properties, paper can serve as the mold for natural or synthetic cell-adhesive hydrogels. Paper can be used as received, or without introduction of hydrophobic walls to define the wells. Upon wicking into the paper, the size and the thickness of the hydrogel is dictated by that of the paper (see, e.g.,
In some instances, the three-dimensional cellular arrays are made using patterned substrates, e.g., patterned paper. Because liquids and gels can readily wick into paper substrates, patterning the paper with liquid impermeable (hydrophobic) borders can be used to dictate not only the physical dimensions but also the shape of the cell growth substrate. Accordingly, in some embodiments, the substrate is patterned into hydrophobic and hydrophilic regions. Any method of patterning the hydrophilic substrate can be used. By way of example, the hydrophobic layer can be applied directly to the porous substrate to produce patterned regions having hydrophilic or hydrophobic properties using printing, such as from an ink jet printer, liquid transfer, such as in stamping or other printing methods, or silk screening. The hydrophobic pattern can also be made using photolithography, in which the paper is infused with photoresist and then exposed to light to produce regions of hydrophobic photoresist and regions of hydrophilic resist-free paper. Exemplary methods are known in the art and described in, e.g., WO 2008/049083, which is incorporated in its entirety by reference.
Exemplary methods for fabricating these patterned paper substrates include a photo-patterning technique, in which a paper is soaked with commercially available photo-reactive polymer (e.g., SU8), and a transparency that presents the desired pattern is overlaid and the paper is briefly exposed to UV light. Upon washing with a suitable solvent, e.g., acetone, the polymer is removed from the exposed areas yielding desired pattern.
Another exemplary method is described herein as “Sweet Patterning”. In certain embodiments, a substrate described herein is patterned using water-soluble compounds, such as sugars or their derivatives (e.g., polyols, e.g., xylitol). These methods stem from the observation that hydrophobic solutions cannot penetrate into regions of paper infused with aqueous solutions. Instead, hydrophobic solvents form complementary patterns within the paper (see, e.g.,
In some embodiments, the substrate is contacted with the water-soluble compound by, e.g., spotting, printing, drawing, or stamping. In a patterning strategy based on conventional inkjet printing, the inverse of a desired hydrophobic pattern is created by printing sucrose solution onto the paper. The paper is then immersed into a solution of polystyrene (or other polymer) that fills the sucrose-free regions. Upon washing with water, the sucrose template is removed and desired hydrophilic-hydrophobic pattern is obtained.
Although the schematic in
Any of a number of known techniques can be used to apply the water-soluble compound to the substrate to produce hydrophilic templates. For example, an aqueous solution of a water-soluble compound can be applied by spotting, printing, drawing, or stamping (see, e.g.,
The methods described herein can utilize any water-soluble compound. These can include, e.g., sucrose, trehalose, glucose, fructose, xylitol, ribose, threitol, mannose, glycerol and other water soluble carbohydrates and their derivatives at least as soluble as those listed above. The appropriate concentration of a water-soluble compound to use on a given substrate and with a given hydrophobic material can be determined using, e.g., the assay described in Example 1 herein. In some embodiments, the water-soluble compound can be insoluble in non-polar organic solvents. In some embodiments, the water-soluble compound is an inorganic salt.
The patterning techniques based on general phase separation described herein can be used to pattern many types of hydrophobic materials onto the paper. For example, the hydrophobic material can be PDMS, poly(lactic-co-glycolic acid), epoxy or polystyrene. Other hydrophobic materials that can be used include, without limitation, any plastic that can be soluble in organic solvents (e.g., polystyrene and derivatives, polyethers, polyamides, PMMA, polycarbonate, polyethylene, polypropylene, photoresist precursors (e.g., SU8), waxes and fats), and/or that can be made by, e.g., polymerization of polycondensation from organic solvents at, e.g., 20-70° C. (e.g., PDMS, polyurethane and epoxy derivatives, phenol-formaldehyde polymers or acrylate and matecrylate derivatives).
HydrogelsA cellular array described herein can be produced by contacting a substrate with a hydrogel or hydrogel precursor, which wicks into the hydrophilic substrate. In certain embodiments, a hydrogel is formed after a precursor has been applied to the substrate. The hydrogel is used with both patterned and unpatterned porous substrates as described herein.
Any known hydrogel can be used in the methods described herein. Hydrogel matrices are described, for example, in U.S. Pat. No. 5,906,934; Lin et al., Advanced Drug Delivery Rev. 58: 1379-1408 (2006); and Jen et al., Biotechnology and Bioengineering 50: 357-364 (2000). Polymers that can form ionic or covalently crosslinked hydrogels that are malleable can be used in the methods described herein. A “hydrogel”, as used herein, is a substance formed when a polymer is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. The polymer can be an organic polymer. The polymer can be a natural or synthetic polymer. As used herein, a “hydrogel precursor” is a polymer that can be cross-linked via covalent, ionic, or hydrogen bonds to form a hydrogel.
Examples of materials that can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates (which are crosslinked ionically) or block copolymers such as Pluronics™ or Tetronics™, polyethylene oxide-polypropylene glycol block copolymers (which are crosslinked by temperature or pH, respectively). Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen. In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
Nonlimiting examples of polymers with acidic side groups that can be reacted with cations include poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate), sulfonated polymers, such as sulfonated polystyrene. Other nonlimiting examples include, e.g., alginic acid (AA), carboxymethylcellulose (CMC), ι-carrageenan, poly(galacturonic acid) (PG), poly(acrylic acid) (PAA), and poly(bis(4-carboxyphenoxy)-phosphazene. Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used. Nonlimiting examples of acidic groups include carboxylic acid groups, sulfonic acid groups, halogenated alcohol groups, phenolic OH groups, and acidic OH groups.
Nonlimiting examples of polymers with basic side groups that can be reacted with anions include poly(vinyl amines), poly(vinyl pyridine), poly(vinyl imidazole), and some imino substituted polyphosphazenes. The ammonium or quaternary salt of the polymers can also be formed from the backbone nitrogens or pendant imino groups. Nonlimiting examples of basic side groups include amino and imino groups.
As used herein, a “gelling agent” is any agent that cross-links a hydrogel precursor to form a hydrogel. For example, a gelling agent can cross-link the hydrogel precursor via covalent, ionic, or hydrogen bonds to form a hydrogel. For example, a water soluble polymer with charged side groups can be ionically crosslinked by reacting the polymer with an aqueous solution containing a gelling agent of opposite charge, e.g., a multivalent ion of the opposite charge. In some embodiments, the polymer has acidic side groups and the gelling agent is a multivalent cation. In other embodiments, the polymer has basic side groups and the gelling agent is a multivalent anion. In some embodiments, the gelling agent is a cation, e.g., Pb2+, Ba2+, Fe3+, Al3+, Cu2+, Cd2+, Ho3+, Ca2+, Zn2+, Co2+, Ni2+, Mn2+, or Mg2+, Sr2+, Gd3+, Pb2+, Ra2+, Fe2+, Pd2+, Bi3+, Hg2+, Au3+, Co2+, Co3+, Cr2+, Cr3+, Mn4+, Pt2+, Pt4+, Sn2+, Sn4+, Ce3+, Ce4+, Ga3+, V3+, or Rh3+.
In other embodiments, the hydrogel is a temperature-sensitive hydrogel (such as Matrigel or collagen), and gelation is induced by raising the temperature of the substrate to an appropriate level (such as 37° C.). The temperature can be maintained by immersing the substrate within a solution at the appropriate temperature, e.g., in a culture medium suitable for a particular type of cell. Temperature-sensitive hydrogels are known in the art and available commercially.
CellsAn array described herein can be loaded with cells simultaneously with a cation solution and/or hydrogel polymer. In some embodiments, the array is loaded with cells after the cation solution and/or hydrogel polymer is contacted with the substrate.
Cells that can be grown in the paper-based cellular arrays can be any prokaryotic or eukaryotic cell. Such cells include, for example, bacterial cells (such as E. coli), insect cells, yeast cells, or mammalian cells (such as Chinese hamster ovary cells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplary cells include cells from the members of the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia, or Streptomyces. Other cells include CD90+/CD45− hepatic tumor stem cells. In certain instances, the cells can be transformed or transfected with one or more expression vectors or viral vectors.
Multilayered Cellular ArraysIn certain embodiments, multi-layer paper-based cellular arrays can be fabricated. Such arrays can be produced by stacking layers of any three-dimensional cellular arrays described herein. For example, two or more paper arrays, each permeated with cell-containing hydrogels, can be stacked to produce a multi-layer array. In one or more embodiments, one or more paper arrays containing cell-free hydrogels can be overlaid with one or more cell-containing paper arrays to arrive at a multi-layer array. In one or more embodiments, one or more of the layers can contain an agent, e.g., a chemical agent, e.g., a chemoattractant. For example, a number of hydrogel-containing sheets can be stacked on top of a chemoattractant-containing sheet, and a cell-containing sheet can be placed on top of the stack.
AssaysAny cell-based assay known in the art can be performed using the three-dimensional cellular arrays described herein. For example, the three-dimensional cellular arrays described herein can be used in screening for agents that influence cell function, such as cell viability, apoptosis, proliferation, migration, and gene expression. Test agents can be added to the cellular arrays, can be adsorbed or covalently attached to the hydrogel or substrate (e.g., paper), or can be also included in a biodegradable matrix coated on the substrate (e.g., paper).
In some instances, a three-dimensional cellular array is contacted with a test agent and the cellular characteristic of interest, e.g., viability, apoptosis, proliferation, migration, or gene expression, is determined using, e.g., an assay described herein. A change in the cellular characteristic of interest in the presence of the test agent relative to a control (e.g., the absence of the test agent) is indicative that the test agent modulates cellular function.
In certain situations, the test agents are contained within wells, e.g., wells of a microtiter plate, and a three-dimensional cellular array is contacted with the test agents in the wells. For example, the test agents can be present in culture medium within the wells of a microtiter plate, and the cellular arrays can be contacted with, e.g., submerged into, the culture medium containing the test agent. In some instances, cellular characteristics can be measured before and after contacting the cellular array with the test agent. In some embodiments, a cellular array can be cut into segments (e.g., strips, cubes, disks, spheres), and a segment can be placed in each well of a microtiter plate.
Multi-layer paper-based arrays can be utilized for analyzing various cellular characteristics, such as migration, assembly, and proliferation. For example, stacked layers can be used for liquid guidance in three dimensions (Martinez et al., Proc Natl Acad Sci U S A. 105:19606-11 (2008)).
In some embodiments, stacks of paper-based arrays can be used as a platform for migration assays. Diffusion gradients of oxygen and nutrients can be established in the multi-layer stack system. Hence, the same assay format provides a convenient system to model the environment in the interior of the solid tumors. In some embodiments, sheet-to-sheet migration of endothelial cells, progenitor cells (EPCs) (Asahara et al., Science 275, 964-967 (1997)), and breast cancer cells towards angiogenic factors, towards nutrients, oxygen or towards a chemoattractants can be investigated. For example, stacking of sheets that contain breast cancer cells in Matrigel and sheets that contain Matrigel without cells can be used to investigate invasion of cells into “blank” Matrigel containing sheets. Specific order of cell containing and “blank” stacks can be chosen to study direction of such migration (see
In other embodiments, hypoxia-induced proliferation, differentiation or apoptosis of cells located at different depths (layers) can be easily quantified when stacked sheets are disassembled. Both migration and hypoxia assays can be used in high-throughput screening.
In particular embodiments, upon destacking, the cells in the separated stacks of paper remain viable and they can be cultured separately or characterized using any assays described herein to compare the cells in separate stacks of paper. For example, viable cells in each sheet can be quantified, using cell proliferation reagents (e.g., Alamar Blue) or fluorimetric assays (e.g., calcein stain,
In some embodiments, co-culture of cells in stacked sheets of paper can be used to investigate self-assembly of tissue-tissue interfaces, cross-migration of cells, and hypoxia responses in a tissue-like environment. For example, three-dimensional cell cultures on paper can be used to investigate the interaction of cells with surrounding tissues, which can be critical for vascular network formation and delivery of nutrients to organs and tumors. The microenvironment formed by endothelial cells can also be examined, which is also critical for proliferation and differentiation of multiple cell types.
Paper arrays that contain cells can be stacked with arrays that contain other cell types to produce a co-culture (see, e.g.,
In another example, endothelial progenitor cells (EPCs) can be cocultured with endothelial cells and breast cancer cells in multi-layered three-dimensional cellular arrays. EPCs in vivo are known to interact with vascular endothelium and undergo transendothelial migration as a first step of homing to ischemic or injured tissue. Conversely, EPC-secreted factors enhance vascularization and promote migratory response in pre-existing endothelial cells (Urbich, et al., Circ. Res. 95:343-353 (2004)). EPCs can be plated in paper-based matrices and then stacked with layers that contain endothelial cell and breast cancer cells to investigate long-term responses in endothelial-EPC co-culture. Cross-migration of these cells in response to angiogenic factors and oxygen concentration gradient can be investigated. Expression profiling and flow cytometry analysis of the EPC and co-cultured cell types can be used to characterize their differentiation state (
In other instances, “multistacks of bacteria” can be used to analyze bacteria grown in various conditions. For example, multilayer stacks of bacteria can be used as a model for bacterial biofilms, which are three-dimensional structures containing multiple layers of bacteria. Bacteria in various layers of a biofilm are exposed to different conditions, such as different amounts of nutrients or oxygen. Bacteria grown in paper and stacked can be used to model the nutrient/oxygen rich and restricted regions present in biofilms. Analysis of the biochemical composition of bacteria in different layers can be used to understand the transformation of bacteria that occur during biofilm formation. It is known in the art that oxygen/nutrient starvation within biofilms can potentially induce bacteria to acquire specific phenotypes that exhibit more or less susceptibility to various cytotoxic agents (K. Lewis, Nat Rev. Microbiol. 2007, 5, 48-56). Thus, analyzing bacterial survival in different layers of a multi-layer cellular array upon treatment with different test agents can be useful to identify agents to treat bacterial biofilms.
One exemplary system is illustrated in
The three-dimensional cellular arrays described herein can be used for high-throughput screening methods, for example, to screen for modulatory agents. In some embodiments, patterned paper (e.g., patterned into hydrophilic and hydrophobic regions) can be spotted with one or more potential modulatory agents. The patterned paper can then be overlayed with a three-dimensional cellular array, the modulatory agent(s) can diffuse into the array, and the effect of the modulatory agents on the cells within the array can be assessed.
As depicted in
In other embodiments, a high throughput screen includes a multi-layer paper-based array. For example, a sheet of paper patterned with small molecules can be stacked onto a number of sheets of paper, one of which is a three-dimensional cellular array. The effect of the small molecules on a cellular property, such as cell migration, can be assayed. An exemplary method is depicted in
In some embodiments, high-throughput screening can be performed by placing cell-containing three-dimensional paper arrays into microtiter plates. Each well of the microtiter plate can include an agent to be screened, and the responses of the cells within the arrays can be assayed.
One exemplary method is depicted in
Another exemplary method is depicted in
Sheets 1840 and 1850 designated “wH” and “woH” above can be used to create screening arrays without limitation to the size, shape, or features of the plate; to the size, shape, or number of holes (wells); or to the materials and methods used to prepare the plate. These sheets can be made by injection molding, casting, machining, laser cutting, or vacuum sheet forming one or more resins. The sheets can be made from transparent or opaque materials; material can be metal, platic, glass, ceramic and other water-impermeable material that is, preferably, non-toxic to cells. In particular embodiment, sheet 1850 designated “wH” can contain an ordered array or holes to create an ordered array of well that can be recognized by instruments which are designed to work with standard microwell plates (e.g., 12×8 array of holes in “wH” can be used to create an array that has a layout identical to 96-well plate). In a particular embodiment, the sheet 1850 designated “wH” with specific patterns of holes can be obtained as commercially available bottomless 96-, 384-, 1536-, and 3456-well plates (e.g., from Greiner Labortechnik of Frickenhausen, Germany; and Corning Life Sciences of Acton, Mass.).
Any of the multi-layer assays described herein can be adapted to be used with a bottomless microtiter plate. For example, multiple layers of patterned paper can be stacked and contacted with “wH” and “woH” sheets (as depicted in
In particular embodiments, three-dimensional cellular arrays are used in a high throughput screening using normal or malignant cells and modulatory agents to identify those that promote (or inhibit) cell death, proliferation, migration or differentiation. If multiple layers of paper are used as described herein, cell death, proliferation, migration or differentiation in different layer in response to modulatory agent can be assessed.
In other embodiments, three-dimensional cellular arrays are used for high throughput screening using progenitor and stem cells. To date, high-throughput screening of stem and progenitor cells has been performed on two dimensional substrates, and these methods can be extended using the cellular arrays described herein. Three-dimensional layers of cells inside the paper arrays mimic the three-dimensional aggregates of cells that are commonly used for growth or differentiation of progenitor and stem cells. For example, three dimensional aggregate of embryonic stem cells (ESCc) termed “embryo bodies” are used for differentiation of ES cells, three dimensional aggregates of neural stem cells (NSCs) termed “neurospheres” are used for proliferation and differentiation of neuronal stem cells. In particular embodiment, cellular arrays can be plated with ESCs or NSC and one or multiple layers of these arrays can be used to create an array of three-dimensional structure similar to “embryo bodies” or “neurospheres”. Differentiation inside such structures can be investigated when the layers of the array are separated. Screening for “modulatory agents” that regulate this differentiation can be performed using any of the methods described herein.
High-throughput Screening of 3D Cultures of Different GeometryPaper-supported arrays can be used in a screening procedure that involves rapid generation of 3D cultures of different geometries and investigation of how the properties of the cells (e.g., metabolic activity, growth, migration, differentiation) are influenced by 3D geometry. Different geometries can be exemplified by (but not limited to): (1) 3D cultures of physical different size or shape. These cultures can be generated by stacking different number of sheets and sheets that contain holes of defined size in defined location. Planar arrangement of holes and vertical arrangement of sheets determines the resulting 3D size and shape of the culture (see, e.g.,
The three examples above are independent and can be used as a combination. For example, a stack of perforated papers can present some areas that have different mechanical properties, and can also include some areas that have cells encapsulated in hydrogels of different composition.
Types of Cellular AssaysThe three-dimensional arrays described herein can be used to characterize various properties of cells grown within the arrays. For example, qualitative and quantitative detection of cells in paper based arrays is possible, such as using fluorescent microscopy or colorimetric assays known in the art.
Certain embodiments will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are known to those of ordinary skill of the art. Such techniques are described in, e.g., “Molecular Cloning: A Laboratory Manual”, third edition (Sambrook et al., 2001); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994).
Apoptosis AssaysAny standard assay for measuring apoptosis known in the art can be used to determine the apoptosis of cells in three-dimensional cellular arrays described herein. Such assays include, without limitation, the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay (Lazebnik et al. 1994, Nature 371, 346); the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747); acridine orange staining (Lucas, R., et al., 1998, Blood 15:4730-41); the caspase-3/7 assay (available as Apo-ONE™ Homogeneous Caspase-3/7 assay from Promega, cat# 67790); and the cell death nucleosome ELISA assay (available from Roche, Cat# 1774425).
In some instances, a test agent can be added to a three-dimensional cellular array described herein and changes in induction of apoptosis relative to controls (where no test agent is added) can be used to identify candidate agents to modulate apoptosis.
Cell Proliferation and Cell Cycle AssaysThe proliferation of cells within three-dimensional cellular arrays described herein can be assayed using any method known in the art. Known methods include, without limitation, bromodeoxyuridine (BRDU) or 5-ethynyl-2′-deoxyuridine (EdU) (
The level of gene expression can be analyzed using the three-dimensional cellular arrays described herein. For example, cells can be lysed directly within a cellular array and the lysates can be used in standard assays known in the art, e.g., Northern analysis, ribonuclease protection assays, or reverse transcription-polymerase chain reaction (RT-PCR) (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001)).
Changes in gene expression in cells grown in three-dimensional arrays can be assayed using known genome-wide analysis techniques. For example, an Affymetrix GeneChip® can be used to perform transcriptome analysis; Illumina Deep Sequenceing can be used to perform the analysis of coding and regulatory RNAs (e.g. micro RNA or miRNA) Gene expression and regulatory RNA profiles can be compared to known profiles for cells grown ex vivo and in vivo.
The cellular material obtained from the cells cultured in three-dimensional cellular arrays can also be used to analyze protein levels, by methods such as by Western analysis or immunoassays. Proteins, carbohydrates and metabolites from the cellular material can be analyzed by known global profiling methods such as those based on mass spectrometry (e.g. shotgun proteomics, metabolomics) or NMR spectroscopy (metabonomics).
Analysis of Isolated CellsThe cells can be isolated from the three-dimensional cellular arrays described herein and used in subsequent assays. For example, isolated cells can be used in flow-cytometry analysis. Cells can be isolated from the arrays using any known method, e.g., enzymatically. One exemplary enzyme is reactive to cellulose substrates, such as cellulase from Trichoderma reesei.
Responses to External StimuliCells grown in three-dimensional cellular arrays can be evaluated for response to known effectors, e.g., effectors of proliferation and morphogenesis. In one exemplary assay, the responses of endothelial cells (e.g., tube and lumen formation) can be evaluated. These responses are known to be regulated by Integrin-ECM interactions and the downstream Rho GTPase-mediated pathways; and a panel of small-molecules and siRNA known to inhibit these processes can be readily obtained and tested (Koh, et al., J. Cell Sci. 121:989-1001 (2008); Ghosh, et al., Proc. Natl. Acad. Sci. U. S. A. in press, doi: 10.1073/pnas.0800835105 (2008)).
Test AgentsThe three-dimensional cellular arrays described herein can be used to assay any test agent. A “test agent” can be any agent, such as a small organic or inorganic molecule, amino acid, polypeptide, nucleic acid, peptide nucleic acid, carbohydrate, or polysaccharide. The test agents can be synthetic, naturally occurring, or a combination of synthetic and natural components. In some embodiments, the test agent can be a member of a library of test agents (e.g., a combinatorial chemical library) or a component of a cellular extract or bodily fluid (e.g., urine, blood, tears, sweat, or saliva).
The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.
EXAMPLES Example 1 Morphology and Growth Rates of Cells Within Matrigel-Permeated PaperThe long-term growth rates of several cells grown within three-dimensional matrices of paper permeated with Matrigel were tested. These cells included primary cells (human umbilical vein endothelial cells (HUVEC), human dermal fibroblasts (HDF), IMR-90 human lung fibroblasts), immortalized cells (telomerase-transfected GFP-HUVEC, HS-5 human bone marrow stroma cells, S16 rat Schwann cells) and cancer cells (MDA-MB-231 human breast adenocarcinoma and PC-12 rat pheochromocytoma). (
The population doubling time of most of the cells in paper-supported Matrigel matrix was significantly lower than their doubling time in 2D culture. The doubling time was the highest for S16 cells (˜36 hours) and PC-12 cells (˜48 hours), while both primary HUVECs and immortalized GFP-HUVEC cell lines exhibited little to no proliferation. These results agree with observations that many cell types suspended in three-dimensional hydrogels proliferate slower than the same cells plated on two-dimensional surfaces of the culture dishes.
Confocal microscopy was used to examine the morphology of cells in paper-supported Matrigel matrices. Human umbilical cord vascular endothelial cells (HUVECs) were used as a model system. HUVECs exhibit distinct morphological change in three-dimensional-cells form hollow structures reminiscent of capillary tubes in vivo. In contrast, HUVECs form flat cell monolayer when cultured on two-dimensional culture dishes. These cells are known to decrease their growth rate when encapsulated inside the three-dimensional matrices.
A suspension of HUVEC in Matrigel were spotted onto paper. In 7-12 days the cells formed hollow blood vessel-like structures. This observation confirmed that cells cultured in Matrigel-permeated paper exhibit behavior characteristic of cells grown in three-dimensional matrices. Fibroblasts, stroma cells and Schwann cells spread out through the cellulose fibers, forming multilayer structures upon long-term proliferation. MDA-MB-231 cells exhibited little spreading and formed disorganized aggregates of cells. PC-12 cells cultured in paper permeated with Matrigel were induced to differentiate into of neuron-like cells that formed three-dimensional network of interconnected neurites.
Overall, paper did not have detrimental effects on the morphology and physiology of the primary and immortalized cells examined
Example 2 Comparison of Gene Expression in Cells Within Matrigel-Permeated Paper and on Two-Dimensional SubstratesCells grown in paper are different from cells grown in two dimensions. Global gene expression profiling was used to investigate how similar cells grown in three dimensions on paper are to cells grown in three dimensions.
Gene expression levels in HUVECs proliferated on chemically identical two-dimensional and three-dimensional systems were compared. Cells cultured on two-dimensional Matrigel monolayers were compared to those cultured inside Matrigel-permeated paper. Cells were lysed and processed to isolate total RNA. Seven candidate genes were analyzed for differential expression in cells grown in two-dimensional and three-dimensional systems.
Quantitative real-time PCR demonstrated that 4 out of 7 selected genes had different expression levels (see
Paper can be used to grow cells in three-dimensional and can subsequently be used to screen for responses in three-dimensional that do not occur in two-dimensional (or those responses are different in two-dimensional). To demonstrate this, paper was used as a platform to investigate three-dimensional migration and tube formation of endothelial cells (HUVEC).
A suspension of cells in Matrigel (1 μL, 107 cells/mL) was spotted onto paper, and the paper was immersed in Matrigel. This resulted in 2-3 mm circular patterns that contained cells in three-dimensional-matrix surrounded by cell-free three-dimensional matrix (see
Cell-containing sheets were suspended in endothelial culture media supplemented with 100 ng/mL VEGF (“VEGF”) or 10 μM of small molecule inhibitor of Rho kinase Y27632 (“Y27632”). The samples were fixed and stained with SYTOX at the days indicated in
In another embodiment, endothelial cells are spotted in the middle and breast cancer cells on the outside and co-cultured in three-dimensional. The cell types are differentially-labeled, and lateral cross-invasion of these two cell types are observed (representing a combination of “metastases” and “angiogenesis”).
Example 4 Application of Paper in Cell-based High-throughput ScreeningPaper-supported three-dimensional substrates can be readily utilized with existing high-throughput screening infrastructures. Paper permeated with a cell-hydrogel suspension were distributed to wells of a 96-well microplates. Distribution of paper pieces of the same size were used to control delivery of similar number of cells per well. High-throughput investigation of cellular responses in three-dimensional were performed using luminescence.
Specifically, a simple cell-based model system was used to produce a luminescent readout. Baby hamster kidney (BHK) cells were infected by vesicular stomatitis virus carrying a luciferase reporter pasmid (lux-VSV). Cells infected with this virus produce luciferase.
A suspension of BHK cells in Matrigel was permeated into 2×4 mm pieces of paper. These pieces were distributed to the wells of a 96-well plate, and the cells were incubated with growth media for 24 hours. An increasing titer of lux-VSV was added to the wells. 6 hours later, the media was removed, a solution of luciferin in cell lysis buffer was added to the wells, and the signal was read using a luminescence plate reader.
A linear increase in luminescence was observed corresponding to the linear increase in lux-VSV titer (
We analyzed the growth of the PC-12 rat adrenal pheochromocytoma cell line in Matrigel-permeated paper. PC-12 cells are known to undergo neuronal differentiation upon treatment with nerve growth factor (NGF).
First, we assessed the proliferation of PC-12 cells grown in paper in response to NGF. Suspensions of PC12 cells in Matrigel (1 μL, 5×106 cells/mL) were spotted onto filter paper. The paper was placed in serum-containing media (2.5% fetal bovine serum, 15% horse serum in F12K basal media) or serum-containing media supplemented with 100 ng/mL of nerve growth factor (NGF). Cells within paper-Matrigel matrix were cultured for the indicated times, fixed with formaldehyde, and stained with Alexa Fluor 488-conjugated phalloidin. The paper as scanned using a Typhoon fluorescent gel scanner (see
Cells proliferated in paper supported matrix with population doubling time of 36 hours. Moreover, in serum containing media, NGF had little effect on growth rate of the cells.
Next, we assessed whether PC-12 cells maintained in paper-supported three-dimensional matrices were differentiated into neurons using NGF-treatment. We spotted suspensions of PC12 cells in Matrigel onto paper and suspended the paper in the media supplemented with NGF or with a combination of NGF and the ROCK inhibitor Y27632. Confocal microscopy examination uncovered neuron-like cells with long neurites. The morphology of cells and length of neurites varied when different media/additives were used. For example, after 6 days of culture in the presence of 100 ng/mL NGF, short neurites were developed by cells in serum containing media, longer neurites were developed in serum-free media, and extensive 3D network of interconnected neurites was observed in serum-free media in the presence of 5 μM Y27632 ROCK inhibitor.
To confirm the observed neuronal phenotype, the cells were analyzed by quantitative PCR and were shown to upregulate the neuron specific neurofilament L (NF-L) and neuropeptide Y (NPY) transcripts (
Cells in these matrices were proliferated in serum-containing media or in media supplemented with 100 ng/mL NFG. On day 3 or 4, the samples were processed to isolate mRNA and the level of expression of NF-L or NPY were assessed by quantitative PCR. GAPDH and actin were used as housekeeping controls. ΔΔCT-analysis was performed in the linear range for every primer using GAPDH as a reference. In
We observed no differences in the levels of NF-L and NPY expression induced by NFG-treatment (
Layering of multiple sheets of paper were used to pattern cells in three dimensions and to create complex three-dimensional assemblies of cells. Importantly, the stacked layers were disassembled and the cells in each layer were able to be examined individually (
To compare the behavior of several cell types in multi-layer cultures, 4 μL of primary cells (HUVECs, HDF, IMR-90) or immortalized cells (HS-5) in Matrigel were spotted onto 200 μm-thick chromatography paper. The cells were incubated in the appropriate media for 24 hours (allowing the cells to spread) and were stacked onto 6-8 layers of paper. To create a uni-directional gradient of oxygen and nutrients in a stacked layer, an impermeable layer was placed on the bottom of the stack (
In the HDF and HS-5 stacked cultures, the density of cells in the top layer was similar to that of the cells proliferated for 11 days in the non-stacked paper (
IMR90 and HUVEC cells exhibited decreased proliferation even in the top layer. We hypothesized that this decrease is due to blocking of the access of nutrients from one side of the paper. In free-floating layer where access of nutrients can occur from either side of the paper and the cells are on average 100 μm or less from the bulk solution. With one side of the paper blocked, the average distance to bulk solution effectively doubles and becomes ˜200 μm. This doubling of the thickness might be detrimental for these cell lines that might be more sensitive to oxygen deprivation. In a control experiment, one layer of HUVEC cells was placed on top of 7 layers that contain Matrigel (without cells) and impermeable layer underneath. This configuration effectively blocks access of nutrient and oxygen from one side of the paper. The number of cells in the top (cell containing) layer was less than that in free-floating layer and it was similar to the number of cells in the top stack of 8 layers of HUVECs (not shown).
In all cases, cell density below the first layer (>200 μm depth) was significantly lower that that in non-stacked layer control (dark line,
Confocal imaging revealed distinct morphological changes of cells in stacked layers. Extensive formation of network of hollow lumens occurred in top 5-6 layers (
Stacking of multiple layers of paper-supported hydrogels were used to investigate three-dimensional migration of cells. Additionally, observation of three-dimensional migration of cells from layer to layer can be used to confirm that a continuous three-dimensional hydrogel is formed upon stacking. A simple way to visualize cell migration is to stack alternating layers that contain cells in paper-supported Matrigel and layers that contain Matrigel only. To create this stack, 4 uL of Matrigel and suspension of cells in Matrigel were spotted onto the sheet of filter paper. A spotting pattern was selected that upon folding yields a “4-helix” of cell-containing areas surrounded by Matrigel-containing areas. (
Following nine days of culture, the folded papers were treated with 4% formaldehyde solution, and stained with phalloidin. The stacks were unfolded and the cells were visualized using a gel scanner (
We used a paper containing a pattern of holes to create 3D cultures of different geometry (
We compared stacks presenting 2, 4 ,6 or 8 layers (i.e., 400, 800, 1200 and 1600 micron thick cultures,
Introduction of cavity in 3D cultures increases the overall metabolic activity of cells in the stack and makes cell in the middle of the 3D culture more metabolically active (e.g. compare stack of 4 layers in 18D and the same stack in 18E made of 2+2 layers separated by 800 micron). Because the bottom and the sides of the stack have no access to nutrients and the influx of nutrients and oxygen happens only through the top of the stack, introduction of cavity inside 3D culture does not change effective perfused surface area of the stack (which is on the top). Finally, the number of cells is same or even higher for “separated stacks”. Despite these similarities between normal stack and “stack with cavity” the combined metabolic activity is significantly higher in stack with cavity.
Example 9 Multiple Layers of Paper-supported Hydrogels to Investigate the Viability of Bacterial CellsThe viability of Pseudomonas Aeruginosa strain PA14 cells grown in a stack of 200 micron chromatography paper was investigated. The paper was soaked with a suspension of bacteria, and bacteria were allowed to attach to the paper for several hours. Eight sheets of paper were then stacked (forming an 8-layer stack) and incubated in culture media for 4 hours, 24 hours, or 48 hours. The papers were destacked and the number of dead and live bacteria in each layer were determined using a commercial Live/Dead Bacterial viability Kit, (Invitrogen).
A steady decrease in the number of both live and dead bacteria in the middle of the 8-layered stack was observed. This decrease can be attributable to competing rates of oxygen diffusion and oxygen consumption by bacteria. Although both the top (layer 1) and the bottom (layer 8) of the stack were exposed to media, the media was not stirred, and layer 8 was farthest away from the air/liquid interface. In non-stirred media, there were fewer viable bacteria in layer 8, presumably because less oxygen can access layer 8 compared to layer 1.
Example 10 Development of Sweet Patterning MethodsA. Screening Carbohydrates and Polyols for Ability to Protect Cellulose for Sweet Patterning Methods
A screen was developed to identify compounds that can protect cellulose from hydrophobic solutions. Sucrose was initially tested, as sucrose is a cheap and abundant carbohydrate. Solutions of sucrose in various concentrations were spotted onto a filter paper, then a solution of polymer or polymer precursor in hydrophobic solvent was soaked into the paper (see
To prepare PDMS-modified paper (
Both PDMS and polystyrene formed complementary patterns around the sucrose solutions. Potentially many other types of thermosetting and thermoplastic polymeric materials could be used. These results suggest that any patterns formed by sucrose solutions on the paper can template formation of the complementary hydrophobic patterns within the paper.
Using the screen outlined in
The protective ability of related compounds was quite different, and the protection of cellulose by carbohydrates and their derivatives was concentration dependent (
The protection profiles against PDMS-octane and polystyrene-toluene varied dramatically (see
The miscibility of liquids within paper could not be predicted from phase behavior of these liquids in the absence of paper. Some immiscible liquids became miscible within paper (e.g., glycerol and octane-PDMS solution). There was a sharp decline in protection efficiency upon minute changes in concentration for all compounds. The screen described in
B. Patterning Using Hydrophilic Solvent, Followed by Spotting by Hydrophilic Solutions
If phase-separation is an equilibrium configuration, the order of the addition of solutions should not influence the final state.
Paper was first soaked in a 1:1 solution of PDMS precursors in n-octane. Solutions of sucrose were spotted onto the PDMS-soaked paper, and the paper was incubated at 70° C. for 2 h to cure the PDMS. Within a few seconds, sucrose drops penetrated into the bulk of the paper and displaced the hydrophobic solution (
C. Patterning of Paper with Hydrophobic Materials
Solutions of sucrose were patterned using many existing techniques for patterning of inks solutions onto the paper. In a first method, patterns were formed using inkjet printing with syrup-filled cartridges (see
Printed patterns remained hydrophilic and could be used for liquid guidance (microfluidics) (see
In another method, microfluidic channels were formed using a syrup-filled fountain pen (63 wt % solution of sucrose). Dashed outlines of two channels were printed using a conventional laser printer. The outlines were “filled” using a syrup-filled pen. Paper was immersed into polystyrene solution (10 wt % in toluene) and the excess solution was blotted with clean filter paper. The paper was dried at room temperature for 5 minutes and washed with water. Ink solutions (Amarant Red and Coomasie Brilliant blue) were spotted and allowed to wick into the channels. Laminar flow was observed in these channels (
In yet another method, patterned paper was fabricated by stamping sucrose solutions onto paper. SU8-patterned paper was used as a basis for the stamp (hydrophilic paper areas are marked with the blue dye). The “stamp” was assembled as shown in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
1. -43. (canceled)
44. A cellular system, comprising:
- a plurality of porous, hydrophilic substrates, wherein the hydrophilic substrates are stacked over one another and at least one of the porous hydrophilic substrates comprises at least one defined region comprising a cell-containing hydrogel.
45. The cellular system of claim 44 wherein the substrate is one or more substrates selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film.
46. The cellular system of claim 44, wherein the cells are one or more cells selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells and mixtures thereof.
47. The cellular system of claim 44, wherein the hydrogel comprises a temperature sensitive hydrogel.
48. The cellular system of claim 44, wherein the hydrogel comprises an ionotropic hydrogel.
49. The cellular system of claim 44, wherein one or more of the plurality of substrates comprises hydrogels.
50. The cellular system of claim 44, wherein one or more of the plurality of substrates comprises a cell-containing hydrogel.
51. The cellular system of claim 44, wherein one or more of the plurality of substrates comprises a test agent.
52. The cellular system of claim 44, further comprising living tissue in contact with the cell-containing hydrogel.
53. The cellular system of claim 44, wherein there are a plurality of porous regions positioned in the porous hydrophilic substrate in the form of an array.
54. The cellular system of claim 53, further comprising:
- a bottomless microtiter plate having a plurality of wells disposed over the porous hydrophilic substrate;
- wherein the wells of the microtiter plate and the plurality of porous regions of the porous hydrophilic substrate are positioned so that the plurality of wells are aligned and sealingly joined to the plurality of porous regions to form an individual chamber for each porous region, wherein the wells and porous regions define an array.
55. A cellular system comprising:
- a porous, hydrophilic substrate, wherein the substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary, wherein at least one of the porous regions comprises a hydrogel comprising cells.
56. The cellular system of claim 55, wherein the liquid impervious boundary is selected from the group consisting of poly(dimethylsiloxane), poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, poly(methyl methacrylate), polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax and a fat and mixtures thereof.
57. The cellular system of claim 55, further comprising:
- a bottomless microtiter plate having a plurality of wells disposed over the porous hydrophilic substrate;
- wherein the wells of the microtiter plate and the liquid impervious boundaries of the porous hydrophilic substrate are positioned so that the plurality of wells are aligned and sealingly joined to the plurality of liquid impervious boundaries to form an individual chamber for each porous region, wherein the wells and porous regions define an array.
58. A kit comprising:
- one or more porous, hydrophilic substrates, wherein the substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary; and
- a bottomless microtiter plate having a plurality of wells equal to the number of porous regions in the substrate, wherein the wells of the microtiter plate and the liquid impervious boundaries of the porous hydrophilic substrate are positioned so that the plurality of wells are aligned.
59. The kit of claim 58, further comprising a hydrogel.
60. The kit of claim 58 wherein the porous hydrophilic substrate is selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film.
61. A method of making a cellular system, comprising:
- providing a porous, hydrophilic substrate; and
- in any order,
- contacting a plurality of defined regions of the substrate with a suspension comprising cells and a hydrogel precursor, wherein the cells and the hydrogel precursor saturate the plurality of defined regions of the substrate; and
- contacting the hydrogel precursor with a gelling agent, wherein the gelling agent induces the formation of a hydrogel embedded in the plurality of defined regions of the substrate.
62. The method of claim 61, wherein the hydrogel precursor is a temperature-sensitive hydrogel or an ionotropic hydrogel precursor.
63. The method of claim 61, wherein cells are introduced into the hydrogel precursor before contacting the hydrogel precursor to the substrate.
64. The method of claim 61, wherein the gelling agent is temperature, a solution containing salt, or a chemical cross-linking agent.
65. The method of claim 61, wherein the substrate is one or more substrates selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film.
66. The method of claim 61, wherein the porous, hydrophilic substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary.
67. The method of claim 66, wherein the liquid impervious boundary is one or more selected from the group consisting of poly(dimethylsiloxane), poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, poly(methyl methacrylate), polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax and a fat and mixtures thereof.
68. The method of claim 61, wherein the cells are one or more cells selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells.
69. The method of claim 61, further comprising contacting the system with living tissue.
70. A method of identifying an agent that modifies cellular activity, the method comprising:
- contacting the cellular system of claim 44 or 54 with one or more test agents; and
- detecting one or more cellular activities in the presence of the one or more test agents.
71. The method of claim 70, wherein the cellular system is contacted with the one or more test agents at a plurality of defined regions.
72. The method of claim 71, wherein each defined region is contacted with a different test agent.
73. The method of claim 70, wherein the cellular activity is one or more selected from the group consisting of proliferation, migration, apotosis, differentiation, viability, upregulation of gene transcription, or downregulation of gene transcription.
74. The method of claim 70, wherein the test agent is selected from the group consisting of one or more of a small molecule, amino acid, polypeptide, nucleic acid, carbohydrate, polysaccharide, and metabolite.
75. The method of claim 70, wherein the cells are selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells and mixtures thereof.
76. A method of claim 70, further comprising:
- cutting the cellular system into a plurality of segments, each segment comprising at least one porous region comprising cells and hydrogel;
- contacting each segment with a test agent or a control; and
- detecting one or more cellular activities in the presence of the test agent.
77. A method of measuring cell migration activity, comprising:
- providing a plurality of porous, hydrophilic substrates, wherein the hydrophilic substrates are stacked over one another;
- introducing a first hydrogel comprising cells within the first defined region in a first substrate;
- introducing a second hydrogel free of the cells within the second defined region in a second substrate; and
- allowing the cells migrate from the first defined region to the second defined region, wherein a measure of cell migration activity is determined.
78. The method of claim 77, wherein cell migration activity is determined by destacking the substrates and characterizing the cells in each of the first and second defined regions.
79. The method of claim 77, wherein the cells are bacterial cells, insect cells, yeast cells, mammalian cells or mixtures thereof.
80. The method of claim 77, further comprising introducing a hydrogel comprising cells into defined regions located in a plurality of substrates.
81. The method of claim 80, wherein the cells of the hydrogel are different in different defined regions.
82. A method of patterning a porous, hydrophilic substrate, the method comprising:
- contacting a porous, hydrophilic substrate with an aqueous solution comprising a water-soluble compound, the solution infiltrating the substrate to form a first region of the substrate that is saturated with the solution and a second region that is not contacted with the solution;
- contacting the substrate with a hydrophobic material, the hydrophobic material saturating the second region; and
- removing the water-soluble compound, resulting in a hydrophilic porous region that is bounded by the hydrophobic material.
83. A method of patterning a porous, hydrophobic substrate, the method comprising:
- contacting a porous, hydrophilic substrate with a hydrophobic material, the hydrophobic material saturating the substrate;
- contacting a region of the substrate with an aqueous solution comprising a water-soluble compound, the solution displacing the hydrophobic material from the region; and
- removing the water-soluble compound, resulting in a hydrophilic porous region that is bounded by the hydrophobic material.
84. The method of claim 82 or 83, wherein the water-soluble compound is selected from the group consisting of sucrose, trehalose, glucose, fructose, xylitol, ribose, threitol, mannose, and glycerol and mixtures thereof.
85. The method of claim 82 or 83, wherein the hydrophobic material is selected from the group consisting of poly(dimethylsiloxane), poly(lactic-co-glycolic acid), epoxy and polystyrene and mixtures thereof.
86. The method of claim 82 or 83, wherein the solution is printed using an inkjet printers.
Type: Application
Filed: Mar 27, 2009
Publication Date: May 5, 2011
Patent Grant number: 9829488
Applicant: PRESIDENT AND FELLOWS OF HARVARD COLLEGE (Cambridge, MA)
Inventors: Ratmir Derda (Cambridge, MA), Anna Laromaine Sague (Cambridge, MA), George M. Whitesides (Newton, MA)
Application Number: 12/934,192
International Classification: C40B 30/06 (20060101); C40B 40/02 (20060101); C40B 50/14 (20060101); B05D 5/00 (20060101); C40B 60/14 (20060101);
